US5126275A - Analytical method using tetrazolium salt indicators having a reflectance plateau - Google Patents

Analytical method using tetrazolium salt indicators having a reflectance plateau Download PDF

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US5126275A
US5126275A US07/584,907 US58490790A US5126275A US 5126275 A US5126275 A US 5126275A US 58490790 A US58490790 A US 58490790A US 5126275 A US5126275 A US 5126275A
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alkoxy
hydrogen
alkyl
cyano
alkylthio
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Robert P. Hatch
Jurgen Kocher
Nan-Horng Lin
Scott Ruetten
Klaus Wehling
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Bayer Corp
Precor Inc
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Miles Inc
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Priority to CA002049231A priority patent/CA2049231C/en
Priority to AU83419/91A priority patent/AU635151B2/en
Priority to EP91115071A priority patent/EP0476454B1/de
Priority to ES91115071T priority patent/ES2093664T3/es
Priority to DK91115071.2T priority patent/DK0476454T3/da
Priority to AT91115071T priority patent/ATE144622T1/de
Priority to DE69122824T priority patent/DE69122824T2/de
Priority to JP3265404A priority patent/JP3069170B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/805—Test papers

Definitions

  • the present invention relates to chromogenic tetrazolium salt indicator compounds useful in the determination of reducing substances, particularly nicotinamide adenine dinucleotide (NADH).
  • NADH nicotinamide adenine dinucleotide
  • Tetrazolium salts are well known as chromogenic indicators responsive to reducing substances. Upon reduction, tetrazolium salts are converted into formazan dye products. These indicators have found use in a wide variety of fields, particularly the medical diagnostic field where they have been applied to, among others, cell staining and the determination of analytes in body fluids such as urine, milk, serum, and plasma. Commonly, the determination of body fluid analytes involves an NAD-dependent enzymatic reaction in which NADH is formed as a function of the amount of analyte present in the sample tested. The amount of NADH generated can then be determined by the reductive conversion of an appropriate tetrazolium salt indicator to its formazan dye product.
  • tetrazolium salt indicators are useful in a variety of different product types.
  • One particular type is the reagent strip.
  • This product is a solid state device comprising a paper or other porous carrier matrix which is impregnated or otherwise incorporated with chemical reagents responsive to a particular analyte, for example, glucose or cholesterol.
  • the incorporated reagent system includes a chromogenic indicator which develops color, or changes color, as a function of the amount of analyte in a sample applied to the matrix.
  • the resulting colorimetric response can be observed visually to give qualitative or semi-quantitative readings. Quantitative results can be obtained by reading the reflectance of the matrix surface at one or more defined wavelengths with an appropriate instrument (reflectance meter).
  • the commonly used tetrazolium salt indicators are 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium chloride (MTT), and 2,2',5,5'-tetraphenyl-3,3'-(3,3'-dimethoxy-4,4'-diphenylene) ditetrazolium chloride (NBT). These compounds show maximum absorption (UVmax) in the range of 465-605 nm.
  • UVmax maximum absorption
  • Another shortcoming of the conventionally used prior art tetrazolium salt indicators relates to the evolution of the instrumentation used to measure their colorimetric response. Rapid advancements are being made in developing smaller, less expensive reflectance meters.
  • One of the more costly components of such meters is the optical system which comprises a light source, a filter or other spectral element for selecting or limiting the wavelength of incident or reflect light, and a sensor.
  • Significant cost savings could be realized by eliminating or combining functions of the optical system elements or by using less expensive components, e.g., LEDs as illuminating light sources.
  • commercially available LEDs emit light having a center wavelength that can vary significantly due to manufacturing variances and temperature dependence.
  • the conventionally used tetrazolium salt indicators INT, MTT, and NBT have reflectance spectra which are strongly sloped in the region above their UVmax. Accordingly, without individually calibrating both each instrument, to account for manufacturing variability in the LED, and each test run, to account for variance due to temperature, large errors can be introduced to the assay result.
  • Tanaka et al Japanese Kokai Tokkvo Koho JP 61000084 (Chem. Abst. 104:203469y) describes the detection of glucose using a formazan chelate obtained by the reduction of 2-(2-benzothiazolyl)-3-(carboxyphenyl)-5-phenyl-2H-tetrazolium halide in the presence of nickel (II).
  • Limbach et al German DE 3,247,894 (Chem. Abst. 101:125929v) relates to the use of INT in glucose assays.
  • 2-thiazolyl tetrazolium salts of Formula A below are characterized by a reflectance spectrum exhibiting an extended plateau above about 600 nm, preferably above about 650 nm. Such reflectance plateau confers improved accuracy to analytical assays in which a test sample is contacted with a reagent strip test device comprising a solid carrier matrix incorporated with a test composition comprising (a) reagents which react with said analyte to produce a reducing substance, e.g., NADH, and (b) an indicator compound which is reduced by such reducing agent to produce a chromogenic dye product, and in which the chromogenic dye product is measured by irradiating the carrier matrix with a light source and sensing the reflectance of the carrier matrix by measuring reflected light with a detector element.
  • a reducing substance e.g., NADH
  • the reflectance plateau property of the present compounds is of particular importance in those situations wherein the light which reaches the detector element consists of a narrow wavelength band whose central wavelength is susceptible to variations of over about ⁇ 5 nm, e.g., when using LED light sources.
  • the thiazolyl tetrazolium salts of the present invention are of the formula: ##STR1##
  • R 3 is aryl or styryl
  • R 4 is aryl
  • X is a suitable counteranion.
  • the present compounds can be categorized into four principal groups:
  • R 1 is carboxyl, carbalkoxy, carbaryloxy, carbamoyl, or cyano
  • R 2 is alkyl or chloro
  • R 1 is alkyl or aryl
  • R 2 is carboxyl, carbalkoxy, carbaryloxy, or carbamoyl or cyano, or
  • R 1 is di- or trifluoroalkyl wherein the fluoro substituents are on the carbon adjacent to the thiazolyl residue in the formula, and R 2 is chloro, and
  • Group 4 Compounds - the mono- and diphenyl substituted thiazolyls wherein one or both of R 1 and R 2 are substituted or unsubstituted phenyl, and if only one is substituted or unsubstituted phenyl, the other is hydrogen or alkyl.
  • FIGS. 1-4 show the reflectance spectra of the formazans produced upon reduction of the prior art tetrazolium salts INT, MTT, NBT, and 2-(benzothiazol-2-yl)-3-(1-naphthyl)-5-phenyltetrazolium salt (USSR) at various concentrations of glucose.
  • FIGS. 5-16 show the corresponding spectra for the formazans from particular indicator compounds of the present invention (see list at the beginning of the Table in the Examples section).
  • C 1-4 used to limit a residue, e.g., C 1-4 alkyl, to those forms which contain between 1 and 4 relevant atoms, e.g., carbon atoms, inclusive.
  • Alkyl - linear and branched hydrocarbon residues of the general formula C n H 2n+1 , preferably "lower alkyl” such as the C 1-4 alkyls of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, and tert-butyl, as well as higher alkyls such as n-pentyl, n-hexyl, and the like.
  • lower alkyl such as the C 1-4 alkyls of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, and tert-butyl, as well as higher alkyls such as n-pentyl, n-hexyl, and the like.
  • Alkylamido - the residue --NRC( ⁇ 0)R' wherein R and R', same or different, are alkyl.
  • Alkylthio the residue --SR wherein R is alkyl.
  • Aryl - organic residues derived from an aromatic carbocyclic or heterocyclic ring or ring system by removal of a hydrogen atom attached to such ring or ring system, e.g., phenyl, naphthyl, pyridyl, oxazolyl, quinolyl, thiazolyl, thienyl, and furanyl.
  • Carbamoyl - the residue --C( ⁇ 0)NRR' wherein R and R', same or different, are hydrogen or alkyl.
  • Halo - fluoro, chloro, and bromo.
  • 2-Thiazolyl tetrazolium salts having the defined reflectance plateau property comprise both compounds which are known in the literature as well as novel compounds. Such compounds will find use in the method of the present invention.
  • such compounds are of Formula A having R 1 and R 2 substituents as defined above for the four principal groups.
  • R 1 and R 2 together form a benzo ring to give a benzothiazolyl residue of Formula A1: ##STR2## wherein
  • R 5 and R 6 , or R 6 and R 7 , or R 7 and R 8 together form a benzo or cyclohexyl ring that is unsubstituted or substituted with alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and wherein the others, same or different, are hydrogen, alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfam
  • R 5 , R 6 , R 7 , and R 8 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and the others, if any, are hydrogen.
  • R 1 and R 2 be unsubstituted phenyl or phenyl substituted with, independently, alkoxy, aryloxy (preferably phenoxy), alkyl, amido, alkylamido, arylamido (preferably phenylamido), alkylthio, arylthio (preferably benzthio), amino, carbamoyl, carbalkoxy, carbaryloxy (preferably carbphenoxy), carboxy, cyano, halo, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and if only one is substituted or unsubstituted phenyl, the other is hydrogen, alkyl, or chloro.
  • R 3 and R 4 can vary widely. Essentially any tetrazolium salt of Formula A in which R 3 is an aryl group or a styryl group and R 4 is an aryl group will have the desired reflectance plateau property of the present invention. Selection of particular aryl residues at the R 3 and R 4 positions will, however, influence the intensity (extinction coefficient) of the formazan, and the position (bathochromicity) and flatness of the plateau.
  • R 3 will preferably be selected from: ##STR3## wherein
  • Q is a bond or --CH ⁇ CH--
  • Yl is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, halo, or hydrogen
  • Y 2 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, alkylthio, amino, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido
  • Y 3 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carbaryloxy, carboxyl, cyano, halo, hydrogen, hydroxyl, nitro, sulfo, s
  • R 4 will preferably be selected from:
  • Y 5 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, halo, hydrogen, nitro, or ureido
  • Y 6 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido
  • Y 7 is alkoxy, aryloxy, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, hydrogen, hydroxyl,
  • R 3 is selected from:
  • Y 2 , Y 3 , and Y 4 are each C 1-4 alkoxy
  • Y 4 is hydrogen and Y 2 and Y 3 are both C 1-4 alkoxy or together form methylenedioxy, or
  • Y 2 and Y 4 are both hydrogen and Y 3 is C 1-4 alkoxy, C 1-4 alkyl, C 1-4 alkylamido, alkylthio, C 1-4 alkylthio, carbamoyl, carb(C 1-4 )alkoxy, carboxyl, cyano, halo, hydrogen, nitro, tri(C 1-4 )alkylammonio, or ureido, and
  • R 3 be selected from:
  • Y 5 is hydrogen and each of Y 6 , Y 7 , and Y 8 is C 1-4 alkoxy,
  • Y 5 and Y 8 are both hydrogen and Y 6 and Y 7 are both C 1-4 alkoxy or together form methylenedioxy
  • Y 5 , Y 6 and Y 8 are each hydrogen and Y 7 is C 1-4 alkoxy, C 1-4 alkylamido, C 1-4 alkylthio, carbamoyl, carb(C 1-4 )alkoxy, carboxyl, cyano, hydroxyl, nitro, phenylazo, sulfo, sulfonamido, sulfamoyl, or ureido, (iv) Y 5 is alkoxy or alkyl, Y 6 and Y 8 are both hydrogen, and Y 7 is C 1-4 alkoxy, C 1-4 alkylamido, C 1-4 alkylthio, benzamido, carbamoyl, carb(C 1-4 )alkoxy, carboxyl, cyano, hydrogen, nitro, phenylazo, or ureido,
  • Y 5 and Y 8 are C 1-4 alkoxy, or
  • Y 5 and Y 8 are C 1-4 alkoxy and Y 7 is C 1-4 -alkylamido or benzamido;
  • R 4 be selected from:
  • the present invention also provides novel tetrazolium salts within Groups 1-4 as follows:
  • R 6 , R 7 , and R 8 are all hydrogen and R 5 is alkoxy, aryloxy, alkyl, carbamoyl, carbalkoxy, or carboxy, or
  • R 5 , R 6 , and R 8 are all hydrogen and R 7 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, or
  • R 5 is hydrogen and R 6 , R 7 , and R 8 are independently alkoxy, aryloxy, or alkyl, or
  • R 6 and R 8 are both hydrogen, R 5 is alkoxy, aryloxy, alkyl, carbamoyl, carbalkoxy, or carboxy, and R 7 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, or cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, or
  • R 5 and R 6 together form a benzo ring
  • R 7 is alkoxy, aryoxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxy, cyano, halo, hydrogen, or ureido
  • R 8 is hydrogen, or
  • R 7 and R 8 together form a benzo ring and R 5 and R 6 are both hydrogen, or
  • R 6 and R 7 together form a cyclohexyl ring, R 5 is alkyl, alkoxy, halo, or hydrogen, and R 8 is hydrogen, and
  • R 3 and R 4 are selected from their preferred residues as described above.
  • R 5 , R 6 , R 7 and P8 are hydrogen
  • R 5 , R 6 , and R 8 are hydrogen and R 7 is C 1-4 alkoxy (especially preferred), C 1-4 alkyl, carboxy, or halo
  • R 5 and R 6 together form a benzo ring
  • R 7 is hydrogen or C 1-4 alkoxy
  • R 8 is hydrogen.
  • R 1 is carb(C 1-4 )alkoxy and R 2 is C 1-4 alkyl or chloro
  • R 1 is C 1-4 alkyl or phenyl
  • R 2 is carb(C 1-4 )alkoxy
  • R 1 is di- or tri-fluoromethyl and R 2 is chloro
  • R 3 and R 4 are selected from their preferred residues as described above. Most preferred are those compounds wherein R 1 is di- or trifluoromethyl and R 2 is chloro.
  • both of R 1 and R 2 are unsubstituted phenyl or phenyl para-substituted with the same group selected from alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carbaryloxy, carboxy, cyano, halo, trialkylammonio, and ureido, or
  • one of R 1 and R 2 is unsubstituted phenyl or phenyl para-substituted with alkoxy or aryloxy and the other is hydrogen.
  • R 1 and R 2 are unsubstituted phenyl or one is unsubstituted phenyl and the other is phenyl para-substituted with C 1-4 alkoxy; or wherein R 1 is unsubstituted phenyl or phenyl para-substituted with C 1-4 alkoxy, C 1-4 alkyl, or halo, and R 2 is hydrogen.
  • counteranion The selection of the counteranion will be based primarily on considerations of stability and solubility of the particular tetrazolium salt of interest. In general, one can select from such counteranions as the inorganic anions chloride, bromide, iodide, nitrate, fluroborate, perchlorate, and sulfate, as well as organic anions such as acetate, oxalate, tartrate, and aryl sulfonates (benzene sulfonate, tosylate).
  • inorganic anions chloride, bromide, iodide, nitrate, fluroborate, perchlorate, and sulfate
  • organic anions such as acetate, oxalate, tartrate, and aryl sulfonates (benzene sulfonate, tosylate).
  • Tetrazolium salts are prepared by methods well known in the literature (Hooper, W. D., Rev. Pure and Appl. Chem., 1969, 19, 221; Putter, R., in Methoden der Organischen Chemie, Houben-Weyl-Muller ed., Thieme Verlag: Stuttgart, 1965, Bd. 10/3, p. 633; Nineham, A. W. Chem. Rev., 1955, pp. 355-483).
  • the tetrazolium salts of the present invention are prepared by first reacting a 2-hydrazinothiazole with an aldehyde and then treating the resulting hydrazone with a diazotized aniline. The resulting formazan is then oxidized to the tetrazolium salt by well known methods. Consequently, the synthesis involves three principal starting materials, the aldehyde, the aniline, and the 2-hydrazinothiazole. ##
  • the Group 1 Compounds of the present invention are prepared from unsubstituted 2-hydrazinobenzothiazole (R 1 and R 2 together form an unsubstituted benzo ring).
  • substituted 2-hydrazinobenzothiazoles are prepared from 1-halo-2-nitro arenes (1) by first displacement of the halide with thiocyanate to produce the nitrothiocyanate (2). This is then reduced with reducing agents such as tin, and the resulting aminothiocyanate (3) cyclized to the 2-aminobenzothiazole (4). ##STR10##
  • the 2-hydrazinothiazole (5) is then prepared by reacting hydrazine with the 2-aminothiazole (4) in ethylene glycol at 140° C. (Liu, K-C., Shih, B-J., Arch. Pharm., 1985, 18, 283). ##STR12##
  • the 2-hydrazinobenzothiazolyls required for the various types of the Group 3 Compounds of the present invention are prepared as follows.
  • the 4-carbalkoxy-5-chlorothiazole are prepared by hydrolysis of 4-trichloromethyl-2,5-dichlorothiazole (9) to yield the carboxylic acid (10) (European Patent Publication No. 348,735). Treatment with reagents such as thionyl chloride yield the acid chloride which reacts with alcohols or amines to yield 4-carbalkoxy (11) or N-alkylcarbamoyl (12) thiazoles. Dehydration of 4-carbamoyl-2,5-dichlorothiazole is accomplished by methods well known in the literature to yield 4-cyano-2,5-dichlorothiazole (13). (March, J. Advanced Organic Chemistry Third Edition; John Wiley and Sons: New York, 1985, pp. 932-933). ##
  • the thiazoles, 4-alkyl or aryl-5-carbalkoxy, amido, or cyano-2-substituted thiazoles are prepared by halogenating the appropriate ⁇ -ketoester, amide or nitrile and reacting it with thiourea or thiosemicarbazone (Metzger, J. V., in Comprehensive Heterocyclic Chemistry, Katritzky, A. R., Rees, C. W., ed.; Peragamon: New York, 1985; vol. 6, Part 4 p. 297; Beyer, H., Bulka, E., Z. Chem., 1962, 2, 321-328). ##
  • the 4,5-diphenyl-2-hydrazinothiazoles are prepared by two methods. One is by first forming a benzoin condensation (17) product between two aldehydes (Ide, W. S., Buck, J. S., Org. React., 1948, Vol. 4, 269). Benzoins not available by this method are prepared from the condensation of a phenyl substituted "acyl anion equivalent" with another aldehyde followed by removal of the carbonyl protecting group (Bertz, S., J. Chem. Soc., Chem. Comm., 1980, 17, 831). A specific example is the condensation of the metallated 0-trimethylsilylcyanohydrin (19) with an aryl aldehyde. The product (20) is deprotected using aqueous acetic acid to afford the benzoin product (17). ##STR17##
  • the benzoin products are then converted into the halide (18) using conventional reagents such as thionyl chloride to produce the ⁇ -haloketones (18) which react with thiourea to produce 4,5-diphenyl-2-aminothiazoles (Traumann, V., Liebigs Ann. Chem., 1888, 250, 31, Dodson et al., J. Am. Chem. Soc., 1945, 67, 2442). These can be converted to the 2-hydrazino compounds with hydrazine as described for the benzo examples.
  • the ⁇ -haloketones may be used to directly yield the necessary hydrazone (24) by treating the haloketone with an N-arylthiosemicarbazone (23) (Johne, S., Schaks, A., Hartung, S., Scharf, K.-D., and Nover, L., Pharmazie, 1979, 34, 790).
  • N-arylthiosemicarbazone Johne, S., Schaks, A., Hartung, S., Scharf, K.-D., and Nover, L., Pharmazie, 1979, 34, 790.
  • aldehydes are obtained from commercial sources or can be prepared by methods familiar to one of ordinary skill in the art.
  • aldehydes may be prepared by benzylic oxidation of an arylmethane (March, J., Advances Organic Chemistry Third Edition; John Wiley and Sons: New York, 1985; p. 1079), reduction of an aryl acid chloride (Ibid. p. 396) or aryl acid derivative, [Larock, R. C., Comprehensive Organic Transformations; VCH: New York, 1989; pp. 604-605).
  • Aryl halides may also be used to synthesize aldehydes.
  • a transmetallation reaction produces an arylmetallic species which can be treated with a variety of reagents, such as dimethylformamide, to produce the aldehyde (ibid, p. 681-683).
  • aryl aldehydes, acids, methanes, and halides may be derivatized with a variety of functional groups prior to their transformation into tetrazolium salts. This may be accomplished by aromatic nucleophilic substitution (March, J., Advanced Organic Chemistry Third Edition; John Wiley and Sons: New York, 1985; pp. 576-607), aromatic electrophilic substitution (ibid., pp. 447-511) or heteroatom-directed metallation reactions (Gschwend, H. W., Rodriguez, H. R., in Organic Reactions, John Wiley and Sons: New York, 1979; Vol. 26, 1).
  • the aldehyde piece of the tetrazolium salt contains a phenol or amine, the groups must be protected so that there is not a reaction between these and the diazotized aniline or oxidizing reagent used to prepare the tetrazolium salt.
  • the phenol may be protected by an acid labile group such as dihydropyran (Greene, T. W., Protective Groups in Organic Synthesis, John Wiley and sons: New York; 1981, pp. 87-113) and which is removed by stirring the tetrazolium salt in acidic conditions.
  • an acid labile group such as dihydropyran (Greene, T. W., Protective Groups in Organic Synthesis, John Wiley and sons: New York; 1981, pp. 87-113) and which is removed by stirring the tetrazolium salt in acidic conditions.
  • Aryl amines may be prepared by reduction of the corresponding nitro or azide compound (Larock, P. C., Comprehensive Organic Transformations; VCH: New York, 1989; pp. 412-415 or 409-410), reaction between an arylmetallic compound and an electrophilic nitrogen reagent, (ibid., pp. 399-400), or rearrangement of acyl azides or oxidized amides (ibid., pp. 431-432).
  • electrophilic and nucleophilic aromatic substitution can be used to introduce different functional groups into the aryl amine or synthetic precursor.
  • the present invention takes advantage of the finding that the 2-thiazolyl tetrazolium salt indicators described herein exhibit an extended plateau in their reflectance spectra above about 00 nm.
  • the most preferred indicator compounds of the present invention have a plateau above about 50 nm (i.e., the flatest about 50 nm wide portion begins between 640 and 660 nm). Such a reflectance plateau confers improved accuracy to analytical tests based on the measurement of reflectance from a reagent strip.
  • Reagent strips are known in the art as analytical devices comprising a solid carrier matrix incorporated with a test composition that produces a color change in response to contact with a liquid test sample containing the analyte of interest.
  • test composition in the case of the present invention, comprises (a) a reagent or reagents which react with the analyte to produce a reducing substance, and (b) a 2-thiazolyl tetrazolium salt as described herein which is reducable by such reducing substance to produce a chromogenic formazan dye product.
  • the color response of such reagent strips can be observed visually to give semi-quantitative values, however, quantitative results are obtained by measuring the reflectance of the carrier matrix at a predetermined wavelength. Such measurements involve irradiating the reacted carrier matrix with a light source and sensing the reflectance of the carrier matrix by measuring reflected light with a detector element.
  • tetrazolium salt indicators having a reflectance plateau is used to particular advantage where reflectance from a reagent strip is read using an instrument which is subject to variability in the central wavelength of its optical system (the combination of light source, detector element, spectral control elements, e.g., filters), and other components).
  • Variability in the central wavelength of the optical system can be caused by a variety of factors, for example, variability in the central wavelength of the principal spectral control element such as the illuminating light source or filters.
  • the wavelength of emitted light will typically vary ⁇ 4 nm within an instrument, and up to ⁇ 8 nm between LEDs in different instruments, due to manufacturing variability.
  • LEDs are suceptible to variable central wavelength due to temperature effects as well.
  • broad band light sources are used with filters to provide spectral control of the central wavelength
  • variability within an instrument is typically under 1 nm, however, between instrument variability can be as high as ⁇ 6 nm.
  • the present invention is applicable in those situations where the central wavelength of the light reaching the detector element in the instrument is susceptible to variations in the range of about ⁇ 5 nm.
  • reagent strip for use in the present invention, selection of the carrier matrix, the test reagents which react with analyte to produce the reducing substance, and the method by which such reagents and the tetrazolium indicator are incorporated with the carrier matrix are matters well known in the art of reagent strips.
  • typical carrier matrices are porous and absorbent paper, cloth, glass fiber filters, polymeric membranes and films, and the like.
  • Incorporation methods include impregation of a formed carrier matrix with a solution, suspension, or other liquid form of the test composition, in one or more steps, followed by drying of the matrix; formation of a matrix in the presence of one or more of the components of the test composition, e.g., by casting or layering solutions of film or membrane forming formulations.
  • the tetrazolium salts of the present invention are particularly advantageous in the detection of NADH. Since NADH is produced in enzyme-catalyzed detection reactions specific for various biochemical substances, the present compounds are particularly useful in medical diagnostic tests, such as in the determination of glucose and cholesterol. However, in general other reducing substances besides NADH can also be detected, such as hydrogen sulfide gas, diborane, arsenic hydride, or phosphorus hydride.
  • the diazonium salt is first prepared by cooling a slurry or solution of 8.5 mmol of the amine in 60 mL of 3 N HCl to 5° C. Sodium nitrite (0.70 g, 10.15 mmol) in 5 mL of water is then added dropwise. After stirring for 30 minutes, the mixture added dropwise to a cold (-25° C.) mixture of 8.5 mmol of the hydrazone in 120 mL of 1:1 (v/v) DMF-pyridine. The reaction is not allowed to warm beyond -15° C. during the addition. The mixture is allowed to warm to room temperature while stirring for two hours. Filtration produces the formazan as a black solid. Impurities can be removed by repeated washing with methanol or refluxing the solid in methanol and filtering while hot.
  • Chlorine gas was passed into a mixture of 1093 g (8.19 mol) 2-chloro-4-methylthiazole and 4 liter methylene chloride in a three-neck flask equipped with a stirrer, thermometer, reflux condenser and gas tube, starting at room temperature. After the exothermic reaction died out, the methylene chloride was distilled off under a slow temperature increase with continued introduction of chlorine; the melt was slowly heated to about 160° C. At about 160° C., mostly excess chlorine gas (recognizable by the slight greenish color of the escaping gas) was bubbled in until a gas chromatogram showed almost solely the desired compound 2,5-dichloro-4-trichloromethylthiazole. Total duration of chlorination 40 to 50 hours.
  • the reagent strip consists of a polystyrene handle onto which a single reagent pad is attached.
  • the reagent pad was 0.2 ⁇ 0.2" square and contains reagents allowing for a color change which was instrumentally read when an aliquot of sample containing the appropriate analyte was applied.
  • the dry-phase reagent pad is a solid support composed of cellulosic fibers or a nylon membrane as examples.
  • the reagent pad was impregnated first with a solution of the tetrazolium salt of interest (0.8M/L) and detergent (0.3%) in a solvent such as methanol.
  • the second solution impregnated into the reagent pad contains the following components:
  • test solutions serum, plasma, aqueous
  • analyte concentrations between 0 and 33 mM/L
  • the reflectance spectra of each indicator was measured at 5 nm increments over the wavelength range of 400 to 1100 nanometers.
  • the first compound presented is A.1.a) and is of the Formula A wherein R 1 and R 2 together form an unsubstituted benzothiazolyl ring, R 4 is 4-nitrophenyl, and R 3 is 4-acetamidophenyl; the second compound, A.1.b), has the same R 1 , R 2 , and R 4 substituents as compound A.1.a) but with R 3 being phenyl; and so forth.
  • the reflectance spectrum of tetrazolium salts is understood to be dependent upon the environment in which they are observed or measured.
  • the data below include a measurement of the relative flatness of the flatest portion of the reflectance spectrum at wavelengths greater than 600 nm, which spectrum is generated using a glucose or cholesterol reagent strip prepared as described in Part B above.
  • the relative flatness of the spectrum is expressed in the data in K/S units normalized for the level of analyte detected as defined below.
  • K/S is defined by the equation ##EQU1## wherein R is instrumentally read reflectance units. Percent change in K/S is the change, expressed as a percentage, over a 50 nm range divided by the average of the high and low K/S values over such range.
  • the plateau property of the present compounds shall be understood, for the purposes of this invention, as a percent change in reflectance spectrum (expressed in terms of K/S as defined in the paragraph above) of less than about 17% over a 30-50 nm wavelength span beginning at a wavelength above about 600 nm.
  • the more preferable compounds exhibit a plateau having a percent change in K/S of less than an about 10% over a 50 nm wavelength span.
  • Most preferred are those tetrazolium salt indicators exhibiting a percent change in K/S of about 5% or less over a 50 nm wavelength span.
  • FIGS. 1-4 show the reflectance spectra of the formazans produced upon reduction of the prior art tetrazolium salts INT, MTT, NBT, and USSR at various concentrations of glucose.
  • FIGS. 5-16 show the corresponding spectra for selected compounds of the present invention. The presence of a plateau in the spectra of the formazans from the present compounds, and its absence from that of the formazans from the prior art compounds is readily apparent.
  • UV The wavelength in nanometers of maximum reflectance peak in the UV reflectance spectrum of the formazan.
  • the extinction coefficient and solvent used during measurement are given in parentheses.
  • nm The position of the flatest portion of the reflectance spectrum of the formazan over a 50 nm wavelength span (expressed as the beginning and ending wavelengths in nanometers).
  • K/S The percent change in K/S units over the above mentioned flatest 50 nm portion of the reflectance spectrum.
  • concentration of analyte used to generate the reflectance spectrum is given in parentheses.
  • Mmol refers to the concentration in mmol/liter.
  • R 3 phenyl UV: 540 nm (4.79 ⁇ 10 3 , dioxane);
  • R 3 4-methoxyphenyl UV: 576 nm (3.89 ⁇ 10 3 , dioxane) nm: 650-700 nm; K/S: 7.5% [11.1 mmol)
  • R 3 4-methoxystyryl UV: 514 nm (7.17 ⁇ 10 3 , dioxane)
  • nm 665-735 nm; K/S: 10% (11.1 mmol) 1602, 1500, 1451, 1259, 1122, 1034, 990, 815, 771 cm -1
  • UV 520 (11 ⁇ 10 3 , water)
  • R 3 4-(2-(2-(2-ethoxy)ethoxy)ethoxy) phenyl (LNH50)
  • IRKBr 2942, 1612, 1504, 1464, 1451, 1261, 1178, 1126, 1061, 837, 658 cm -1
  • UV 500 (5.10 ⁇ 10 3 , water)
  • UV 500 (6.18 ⁇ 10 3 , water)

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CA002049231A CA2049231C (en) 1990-09-19 1991-08-15 Analytical method using tetrazolium salt indicators having a reflectance plateau
AU83419/91A AU635151B2 (en) 1990-09-19 1991-08-27 Analytical method using tetrazolium salt indicators having a reflectance plateau
AT91115071T ATE144622T1 (de) 1990-09-19 1991-09-06 Analytisches verfahren unter verwendung von tetrazoliumsalzindikatoren mit reflektionsplateau
ES91115071T ES2093664T3 (es) 1990-09-19 1991-09-06 Procedimiento analitico que usa indicadores de sales de tetrazolio que tienen una meseta de reflectancia.
DK91115071.2T DK0476454T3 (da) 1990-09-19 1991-09-06 Analysemetode under anvendelse af tetrazoliumsalt-indikatorer med et reflektansplateau
EP91115071A EP0476454B1 (de) 1990-09-19 1991-09-06 Analytisches Verfahren unter Verwendung von Tetrazoliumsalzindikatoren mit Reflektionsplateau
DE69122824T DE69122824T2 (de) 1990-09-19 1991-09-06 Analytisches Verfahren unter Verwendung von Tetrazoliumsalzindikatoren mit Reflektionsplateau
JP3265404A JP3069170B2 (ja) 1990-09-19 1991-09-18 反射率プラトーを有するテトラゾリウム塩指示薬を用いる分析方法
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WO1994001578A1 (en) * 1992-07-02 1994-01-20 Boehringer Mannheim Corporation Stabilizing tetrazolium salts in a reagent
US5443946A (en) * 1992-06-05 1995-08-22 Fuji Photo Film Co., Ltd. Silver halide color photographic material and method for forming color image
US5565363A (en) * 1991-10-21 1996-10-15 Wako Pure Chemical Industries, Ltd. Reagent composition for measuring ionic strength or specific gravity of aqueous solution samples
US20030068666A1 (en) * 2001-10-05 2003-04-10 Zweig Stephen Eliot Dual glucose-hydroxybutyrate analytical sensors
EP1445329A2 (de) * 2003-02-04 2004-08-11 Bayer Healthcare, LLC Methode und Teststreifen zur Bestimmung von Glukose in Blut
US20050242392A1 (en) * 2004-04-30 2005-11-03 Siliconix Incorporated Super trench MOSFET including buried source electrode and method of fabricating the same
US20060051738A1 (en) * 2001-10-05 2006-03-09 Zweig Stephen E Dual glucose-turbidimetric analytical sensors
US20060226494A1 (en) * 2004-12-27 2006-10-12 Third Dimension (3D) Semiconductor, Inc. Tungsten plug drain extension
US20070275430A1 (en) * 2004-08-24 2007-11-29 Marfurt Karen L Determining The Concentration Of Analytes In Sample By Direct Mediation Of Enzymes
US20090123955A1 (en) * 2004-12-13 2009-05-14 Bayer Healthcare Llc Size Self-Limiting Compositions and Test Devices for Measuring Analytes in Biological Fluids
US7767822B2 (en) 2005-01-14 2010-08-03 Bayer Healthcare Llc Water-soluble tetrazolium salts
US9515137B2 (en) 2013-02-21 2016-12-06 Infineon Technologies Austria Ag Super junction semiconductor device with a nominal breakdown voltage in a cell area
US10329684B2 (en) 2007-12-10 2019-06-25 Ascensia Diabetes Care Holdings Ag Method for forming an optical test sensor

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GB2261729A (en) * 1991-10-11 1993-05-26 Ashutosh Sharma Method and optical probe for the determination of reduced nicotinamide adenine dinucleotide in a sample
JP3589313B2 (ja) * 1994-11-29 2004-11-17 明治乳業株式会社 牛乳の評価方法及び評価用キット
US6656697B1 (en) 1998-09-28 2003-12-02 Lifescan, Inc. Diagnostics based on tetrazolium compounds
US5902731A (en) * 1998-09-28 1999-05-11 Lifescan, Inc. Diagnostics based on tetrazolium compounds
JP2000262298A (ja) * 1999-03-15 2000-09-26 Fuji Photo Film Co Ltd 全血中のグルコース濃度もしくはコレステロール濃度の定量方法
DE10351088A1 (de) * 2003-10-31 2005-06-02 Bayer Cropscience Gmbh Verfahren zum Herstellen von fluormethyl-substituierten Heterocyclen
JP6893215B2 (ja) * 2016-09-14 2021-06-23 テルモ株式会社 ２−置換チアゾリル−３−置換フェニル−５−スルホ化フェニル−２ｈ−テトラゾリウム塩、ならびに当該塩を含む生体成分濃度測定用試薬および当該塩を用いる生体成分濃度の測定方法
JP6947736B2 (ja) * 2016-09-14 2021-10-13 テルモ株式会社 ２−置換ベンゾチアゾリル−３−置換フェニル−５−置換スルホ化フェニル−２ｈ−テトラゾリウム塩、ならびに当該塩を含む生体成分濃度測定用試薬および当該塩を用いる生体成分濃度の測定方法

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US5565363A (en) * 1991-10-21 1996-10-15 Wako Pure Chemical Industries, Ltd. Reagent composition for measuring ionic strength or specific gravity of aqueous solution samples
US5443946A (en) * 1992-06-05 1995-08-22 Fuji Photo Film Co., Ltd. Silver halide color photographic material and method for forming color image
WO1994001578A1 (en) * 1992-07-02 1994-01-20 Boehringer Mannheim Corporation Stabilizing tetrazolium salts in a reagent
US5583006A (en) * 1992-07-02 1996-12-10 Boehringer Mannheim Corporation Stabilizing tetrazolium salts in a reagent
US6984307B2 (en) * 2001-10-05 2006-01-10 Stephen Eliot Zweig Dual glucose-hydroxybutyrate analytical sensors
US7758744B2 (en) 2001-10-05 2010-07-20 Stephen Eliot Zweig Dual glucose-turbidimetric analytical sensors
US20030068666A1 (en) * 2001-10-05 2003-04-10 Zweig Stephen Eliot Dual glucose-hydroxybutyrate analytical sensors
US20060051738A1 (en) * 2001-10-05 2006-03-09 Zweig Stephen E Dual glucose-turbidimetric analytical sensors
EP1445329A2 (de) * 2003-02-04 2004-08-11 Bayer Healthcare, LLC Methode und Teststreifen zur Bestimmung von Glukose in Blut
US20040185517A1 (en) * 2003-02-04 2004-09-23 Marfurt Karen L. Method and test strip for determining glucose in blood
EP1445329A3 (de) * 2003-02-04 2005-12-28 Bayer HealthCare LLC Methode und Teststreifen zur Bestimmung von Glukose in Blut
US20090075389A1 (en) * 2003-02-04 2009-03-19 Bayer Healthcare Llc Method for determining an analyte in a fluid
US7439033B2 (en) 2003-02-04 2008-10-21 Bayer Healthcare Llc Method and test strip for determining a fluid analyte
US20080182376A1 (en) * 2004-04-30 2008-07-31 Siliconix Incorporated Method of fabricating super trench MOSFET including buried source electrode
US20070187753A1 (en) * 2004-04-30 2007-08-16 Siliconix Incorporated Super trench MOSFET including buried source electrode and method of fabricating the same
US20100019316A1 (en) * 2004-04-30 2010-01-28 Siliconix Incorporated Method of fabricating super trench MOSFET including buried source electrode
US20050242392A1 (en) * 2004-04-30 2005-11-03 Siliconix Incorporated Super trench MOSFET including buried source electrode and method of fabricating the same
US20070275430A1 (en) * 2004-08-24 2007-11-29 Marfurt Karen L Determining The Concentration Of Analytes In Sample By Direct Mediation Of Enzymes
US7867728B2 (en) 2004-08-24 2011-01-11 Bayer Healthcare Llc Determining the concentration of analytes in sample by direct mediation of enzymes
US7964372B2 (en) 2004-12-13 2011-06-21 Bayer Healthcare Llc Size self-limiting compositions and test devices for measuring analytes in biological fluids
US20090123955A1 (en) * 2004-12-13 2009-05-14 Bayer Healthcare Llc Size Self-Limiting Compositions and Test Devices for Measuring Analytes in Biological Fluids
US9982289B2 (en) 2004-12-13 2018-05-29 Ascensia Diabetes Care Holdings Ag Size self-limiting compositions and test devices for measuring analytes in biological fluids
US20060226494A1 (en) * 2004-12-27 2006-10-12 Third Dimension (3D) Semiconductor, Inc. Tungsten plug drain extension
US7767822B2 (en) 2005-01-14 2010-08-03 Bayer Healthcare Llc Water-soluble tetrazolium salts
US7897331B2 (en) 2005-01-14 2011-03-01 Bayer Healthcare Llc Process of using a tetrazolium salt
US20100255506A1 (en) * 2005-01-14 2010-10-07 Bayer Healthcare Llc Process of using a tetrazolium salt
US10329684B2 (en) 2007-12-10 2019-06-25 Ascensia Diabetes Care Holdings Ag Method for forming an optical test sensor
US9515137B2 (en) 2013-02-21 2016-12-06 Infineon Technologies Austria Ag Super junction semiconductor device with a nominal breakdown voltage in a cell area

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